Cancer-cell proliferation-suppressing material produced by cancer cells restricted by entrapment

ABSTRACT

A material for suppressing proliferation of cancer cells is produced by entrapping cancer cells in a selectively-permeable structure such as a bead, and culturing the entrapped cells in a culture medium. Entrapment restricts growth of the cancel cells during culturing and causes the cells to produce in the culture medium a material having a molecular weight of at least about 30 kd as measured by filtration that suppresses proliferation of cancer cells. The material is separated from the culture medium by filtering the medium through a filter that separates material having a molecular weight of at least about 30 kd from material having a molecular weight of less than 30 kd. The structure that entraps the cells may contain 10,000 to 500,000 cells. The material or the structure containing the entrapped cells that produce the material can be administered to a subject to suppress cancer cell proliferation.

RELATED APPLICATION

This application is a continuation in part of U.S. patent applicationSer. No. 09/188,476 filed Nov. 9, 1998, now U.S. Pat. No. 6,224,912which is a continuation in part of U.S. patent application Ser. No.08/745,063, filed on Nov. 7,1996, now U.S. Pat. No.5,888,497 which is acontinuation-in-part now application Ser. No. 08/625,595, filed Apr. 3,1996, abandoned, each of which are incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the restriction of the proliferation ofcells in or capable of rapid log-phase growth which, when theirproliferation is physically restricted in a biocompatible, semipermeablematerial, produce normally unexpressed or increased amounts of anormally expressed factor or factors capable of inhibiting the growth ofother rapidly proliferating cells of the same or different types and/ororigins. These restricted cells are hereinafter referred to asproliferative cells. A non-limiting list of proliferative cellsbelonging to this category include neoplastic cells, cancer cells, andnon-cancerous cells including, but not limited to, embryonic cells, stemcells, and cells in a post tissue injury regenerative phase includinghepatocytes, fibroblasts and epithelial cells. The structures which areone feature of the invention can be used “as is,” or to produce materialsuch as concentrates with a defined approximate molecular weight, whichalso have an anti-proliferative effect on rapidly proliferating cellsassociated with diseases or conditions such as, e.g., cancer, or tocontrol growth of cells to prevent certain medical problems that mayarise from uncontrolled cell growth, such as the formation ofpost-operative scars.

BACKGROUND AND PRIOR ART

The encapsulation of various biological materials in biologicallycompatible materials, which is well documented in the literature, is atechnique that has been used for some time, albeit with limited success.Exemplary of the art are U.S. Pat. No. 5,227,298 (Weber, et al.); U.S.Pat. No. 5,053,332 (Cook, et al.); U.S. Pat. No. 4,997,443 (Walthall, etal.); U.S. Pat. No. 4,971,833 (Larsson, et al.); U.S. Pat. No. 4,902,295(Walthall, et al.); U.S. Pat. No. 4,798,786 (Tice, et al.); U.S. Pat.No. 4,673,566 (Goosen, et al.); U.S. Pat. No. 4,647,536 (Mosbach, etal.); U.S. Pat. No. 4,409,331 (Lim); U.S. Pat. No. 4,392,909 (Lim); U.S.Pat. No. 4,352,883 (Lim); and U.S. Pat. No. 4,663,286 (Tsang, et al.).Also of note is U.S. Pat. No. 5,643,569 to Jain, et al., incorporated byreference herein. Jain, et al. discuss, in some detail, theencapsulation of islets in various biocompatible materials. Isletsproduce insulin, and the use of the materials disclosed by Jain, et al.in the treatment of diabetes is taught therein. U.S. Pat. No. 5,888,497to Jain et al. describe implantable agarose coated, agarose beads havingcancer cells restricted therein which produce more of a material thatsuppresses the growth of non-restricted cancer cells than an equalnumber of the same unrestricted cancer cells.

The Jain, et al. patents discuss, in some detail, the prior approachestaken by the art in transplantation therapy. These are summarized hereinas well.

Five major approaches to protecting the transplanted tissue from thehost's immune response are known. All involve attempts to isolate thetransplanted tissue from the host's immune system. The immunoisolationtechniques used to date include: extravascular diffusion chambers,intravascular diffusion chambers, intravascular ultrafiltrationchambers, microencapsulation, and macroencapsulation. There are manyproblems associated with methods of the prior art, including a hostfibrotic response to the implant material, instability of the implantmaterial, limited nutrient diffusion across semi-permeable membranes,secretagogue and product permeability, and diffusion lag-time acrosssemi-permeable membrane barriers.

For example, a microencapsulation procedure for enclosing viable cells,tissues, and other labile membranes within a semipermeable membrane wasdeveloped by Lim in 1978. (Lim, Research report to Damon Corporation(1978)). Lim used microcapsules of alginate and poly L-lysine toencapsulate islets of Langerhans (referred to as “islets” hereinafter).In 1980, the first successful in vivo application of this noveltechnique in diabetes research was reported (Lim, et al., Science 210:908 (1980)). The implantation of these microencapsulated islets resultedin sustaining a euglycemic state in diabetic animals. Otherinvestigators, however, repeating these experiments, found the alginateto cause a tissue reaction and were unable to reproduce Lim, et al.'sresults (Lamberti, et al. Applied Biochemistry and Biotechnology 10: 101(1984); Dupuy, et al., J. Biomed. Material and Res. 22:1061 (1988);Weber, et al., Transplantation 49:396 (1990); and Doon-shiong, et al.,Transplantation Proceedings 22: 754 (1990)). The water solubility ofthese polymers is now considered to be responsible for the limitedstability and biocompatibility of these microcapsules in vivo (Dupuy, etal., supra, Weber et al., supra, Doon-shiong, et al., supra, andSmidsrod, Faraday Discussion of Chemical Society 57. 263 (1974)).

Iwata et al., (Iwata, et al. Jour. Biomedical Material and Res. 26: 967(1992)) utilized agarose for microencapsulation of allogeneic pancreaticislets and discovered that it could be used as a medium for thepreparation of microbeads. In their study, 1500-2000 islets weremicroencapsulated individually in 5% agarose and implanted intostreptozotocin-induced diabetic mice. The graft survived for a longperiod of time, and the recipients maintained normoglycemiaindefinitely.

Their method, however, suffers from a number of drawbacks. It iscumbersome and inaccurate. For example, many beads remain partiallycoated and several hundred beads of empty agarose form. Additional timeis thus required to separate encapsulated islets from empty beads.Moreover, most of the implanted microbeads gather in the pelvic cavity,and a large number of islets in completely coated individual beads arerequired to achieve normoglycemia. Furthermore, the transplanted beadsare difficult to retrieve, tend to be fragile, and release islets easilyupon slight damage.

A macroencapsulation procedure has also been tested. Macrocapsules ofvarious different materials, such as poly-2-hydroxyethyl-methacrylate,polyvinylchloride-c-acrylic acid, and cellulose acetate were made forthe immunoisolation of islets. (See Altman, et al., Diabetes 35: 625(1986); Altman, et al., Transplantation: American Society of ArtificialInternal Organs 30: 382 (1984); Ronel, et al., Jour. Biomedical MaterialResearch 17: 855 (1983); Klomp, et al., Jour. Biomedical MaterialResearch 17: 865-871 (1983)). In all these studies, only a transitorynormalization of glycemia was achieved.

Archer, et al., Journal of Surgical Research 28: 77 (1980), used acryliccopolymer hollow fibers to temporarily prevent rejection of isletxenografts. They reported long-term survival of dispersed neonatalmurine pancreatic grafts in hollow fibers which were transplanted intodiabetic hamsters. Recently Lacy, et al., Science 254: 1782-1784 (1991)confirmed their results, but found the euglycemic state to be atransient phase. They found that when the islets are injected into thefiber, they aggregate within the hollow tube with resultant necrosis inthe central portion of the islet masses. The central necrosis precludedprolongation of the graft. To solve this problem, they used alginate todisperse the islets in the fiber. However, this experiment has not beenrepeated extensively. Therefore, the membrane's function as an islettransplantation medium in humans is questionable.

The Jain, et al. patent discussed supra reports that encapsulatingsecretory cells in a permeable, hydrophilic gel material results in afunctional, non-innunogenic material, that can be transplanted intoanimals, can be stored for long lengths of time, and is therapeuticallyuseful in vivo. The macroencapsulation of the secretory cells provided amore effective and manageable technique for secretory celltransplantation.

The patent does not discuss at any length the incorporation of cellscapable of rapid proliferation.

A survey of the literature on encapsulation of cells reveals that,following encapsulation, cells almost always produce less of materialsthan they produce when not encapsulated. See Lloyd-George, et al.,Biomat. Art. Cells & Immob. Biotech. 21(3): 323-333 (1993); Schinstine,et al., Cell Transplant 4(1): 93-102 (1995); Chicheportiche, et al.,Diabetologica 31:54-57 (1988); Jaeger, al., Progress In Brain Research82:41-46 (1990); Zekorn, et al., Diabetologica 29:99-106 (1992); Zhou,et al., Am. J. Physiol 274: C1356-1362 (1998); Darquy, et al.,Diabetologica 28:776-780 (1985); Tse, et al., Biotech. & Bioeng.51:271-280 (1996); Jaeger, et al., J. Neurol. 21:469-480 (1992);Hortelano, et al., Blood 87(12): 5095-5103 (1996); Gardiner, et al.,Transp. Proc. 29:2019-2020 (1997). None of these references deal withthe incorporation of cells capable of rapid proliferation into astructure which entraps them and restricts their growth, but nonethelesspermits diffusion of materials into and out of the structure.

One theory relating to the growth of cancerous masses likens suchmasses, e.g., tumors, to normal organs. Healthy organs, e.g. the liver,grow to a particular size, and then grow no larger; however, if aportion of the liver is removed, it will regenerate to a certain extent.This phenomenon is also observed with tumors. To summarize, it has beennoted that, if a portion of a tumor is removed, the cells in theremaining portion of the tumor will begin to proliferate very rapidlyuntil the resulting tumor reaches a particular size, after whichproliferation slows down, or ceases. This suggests that there is someinternal regulation of cancer cells.

SUMMARY OF THE INVENTION

The invention, which will be seen in the following disclosure, showsthat when cells capable of rapid proliferation are physically restrictedsuch as by being entrapped, their proliferation rate decreases markedly,and they produce unexpectedly high amounts of material which, whenapplied to non-restricted rapidly proliferating cells, inhibits theproliferation of these non-restricted cells. The ability to retardproliferation of cancer cells has been a goal of oncology since itsinception. Hence, the therapeutic usefulness of this invention withrespect to its treatment of cancer and other disease states andconditions caused by rapid cell proliferation will be clear and will beelaborated upon herein. The material produced does not appear to belimited by the type of rapidly proliferating cell used, nor by theanimal species from which the rapidly proliferating cells originate.Further, the effect does not appear to be species specific, asrestricted cells from a first species produce material which inhibitsproliferation of unrestricted cells from a second species. Also, withrespect to cancer, the effect does not appear to be specific to the typeof cancer, as restricted cells from a first cancer type produce materialwhich inhibits proliferation of unrestricted cells from another cancertype.

Nor does the effect appear to require an immune response. Theantiproliferative effect is seen in in vitro systems, where no immunecells are used. Hence the antiproliferative effect cannot be attributedto classical immunological responses.

Thus, a preferred embodiment of the invention relates to a compositionof matter having a biocompatible, proliferation-restrictive,selectively-permeable structure. The structure restricts the rapidlyproliferating cells which then produce more of a material whichsuppresses rapid cell proliferation compared to an equal number of thesame rapidly proliferating cells when unrestricted.

Another preferred embodiment of the present invention relates to aprocess for preparing a biocompatible, proliferation-restrictive,selectively-permeable structure, by forming a structure by contactingrapidly proliferating cells with biocompatable,proliferation-restrictive matter to form the structure, and culturingthe structures for a sufficient period of time to restrict the rapidlyproliferating cells such that they produce a material which suppressesproliferation of the rapidly proliferating cells to be suppressedcompared to an equal number of unrestricted rapidly proliferating cellsof the same type.

Yet another preferred embodiment relates to a method of increasing theproduction of material that suppresses cell growth by rapidlyproliferating cells, comprising restricting rapidly proliferating cellsin a biocompatable, selectively-permeable, proliferation-restrictivestructure and culturing the rapidly proliferating cells so that theyproduce the material. When the structures referred to are placed inculture medium, the material referred to leaves the structure and entersthe culture medium. The resulting culture medium is also a feature ofthe invention.

It has also been found that a powerful antiproliferative effect can beachieved by subjecting to filtration the conditioned medium obtained byculturing the structures of the invention in culture medium. Theresulting concentrates have extremely strong anti-proliferative effects.

The material, the conditioned medium, and/or the concentrates derivedtherefrom may also be useful for inducing the production of theanti-proliferative material by other non-restricted rapidlyproliferating cells.

In preferred embodiments, the restricted cells are cancer cells, but theadvantages of using non-cancerous cells to treat cancer and otherconditions provide additional benefits, readily apparent to thoseskilled in the art.

These, and other features of the invention, will be seen from thedisclosure which follows.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

This example, and those which follow, employ RENCA cells. These arespontaneous renal adenocarcinoma cells of BALB/C mice, which are widelyavailable, having been maintained in both in vitro cultures and in vivo.See Franco, et al., Cytokine Induced Tumor Immunogenecity, 181-193(1994).

Samples of frozen RENCA cells were thawed at 37° C., and then placed intissue culture flasks containing Dulbecco's Modified Medium (D-MEM),which had been supplemented with 10% bovine serum, penicillin (100 u/ml)and streptomycin (50 ug/ml), to give what will be referred to as“complete medium” hereafter.

Cells were grown to confluence, and then trypsinized, followed bywashing with Hank's Balanced Salt Solution, and then with the completemedium referred to supra.

In order to determine if the RENCA cells produced tumors efficiently,two BALB/C mice were injected, intraperitoneally, with 10⁶ of thesecells. The mice were observed, over a 3-4 week period. Clinically, theyappeared healthy for the first two weeks, and exhibited normal activity.Thereafter, the clinical manifestations of cancer became evident. Onemouse died after 23 days, and the second, after 25 days. Followingdeath, the mice were examined, and numerous tumors of various size wereobserved. Some of the tumors exhibited hemorrhaging as well.

A sample of one tumor, taken from one of the mice, was fixed in 10%formalin for later histological examination.

EXAMPLE 2

Following the showing that the RENCA cells did grow in vivo, studieswere carried out to determine if these cells grew when restricted in thestructure of the invention.

RENCA cells were grown to confluency, as described supra, trypsinized,and washed, also as described above. Samples of between 60,000 and90,000 cells were then prepared. The cells were then centrifuged, at 750RPMs, and fluid was removed. The cells were then suspended in solutionsof 1% atelocollagen, in phosphate buffered saline solution, at a pH of6.5.

A 1% solution of low viscosity agarose was prepared in minimal essentialmedium (MEM), maintained at 60° C., and then 100 ul of this was added tothe suspension of RENCA cells and atelocollagen, described supra. Thematerials were then transferred, immediately, as a single large droplet,into sterile, room-temperature mineral oil. The mixture formed a single,smooth, semi-solid bead. This procedure was repeated to produce a numberof beads.

After one minute, the beads were transferred to complete medium, asdescribed supra, at 37° C. The beads were then washed three times inMinimal Essential Medium (MEM) containing the antibiotics listed supra.The beads were then incubated overnight at 37° C., in a humidifiedatmosphere of air and 5% CO₂. Following the incubation the beads, nowsolid, were transferred to a sterile spoon which contained 1 ml of 5%agarose in MEM. Beads were rolled in the solution 2-3 times to uniformlycoat them with agarose. The beads were transferred to mineral oil beforethe agarose solidified, to yield a smooth outer surface. After 60seconds, the beads were washed, five times, with complete medium at 37°C. to remove the oil. Overnight incubation (37° C., humidifiedatmosphere of air, 5% CO₂) followed.

These RENCA containing beads were used in the experiments which follow.

EXAMPLE 3

Prior to carrying out in vivo investigations, it was necessary todetermine if the RENCA cells would grow in beads prepared in the mannerdescribed supra.

To do this, beads prepared as discussed in example 2 were incubated inthe medium described in example 2, for a period of three weeks, underthe described conditions. Three of the beads were then cut into smallpieces, and cultured in standard culture flasks, affording directcontact with both the flask and culture medium.

Observation of these cultures indicated that the cells grew and formedstandard RENCA colonies. This indicated that the cells had remainedviable in the beads.

EXAMPLE 4

In vivo experiments were then carried out. In these experiments, thebeads were incubated for seven days, at 37° C. Subject mice thenreceived bead transplants. To do this, each of four mice received amidline incision, carried through intraperitoneally. Three beads, eachof which contained 60,000 RENCA cells were transplanted. Incisions werethen closed (two-layer closure), using an absorbable suture. The fourmice (BALB/C) were normal, male mice, weighing between 24-26 grams, andappeared to be healthy. Two sets of controls were set up. In the firstset, two mice received three beads containing no RENCA cells, and in thesecond, two mice were not treated with anything.

Three weeks after the implantation, all of the mice receivedintraperitoneal injections of 10⁶ RENCA cells. Eighteen days later, onecontrol mouse died. All remaining mice were then sacrificed, andevaluated for the presence or absence of tumor.

Control mice showed numerous tumors, while the mice which received theimplants of bead-encapsulated cells showed only isolated small nodulesthroughout the cavity.

These encouraging results suggested the design of the experiments setforth in the following example.

EXAMPLE 5

In these experiments, established cancers were simulated by injectingRENCA cells under one kidney capsule of each of six BALB/C mice. Fifteendays later, mice were divided into two groups. The three mice in thefirst group each received three beads, as described in example 4, supra.The second group (the control group) received beads which did notcontain RENCA cells.

For the initial 4-5 days, mice which had received RENCA cell containingimplants looked lethargic, and their fur had become spiky. Thereafter,they returned to normal. The control group remained energetic, with nochange in condition of fur.

Ten days after implantation (25 days after injection of RENCA cells),however, the control mice became sluggish and exhibited distendedabdomens. One of the three control mice died at fourteen days followingbead transplantation. Sacrifice of the mice followed.

The body cavities of the control mice showed profuse hemorrhaging, withnumerous tumors all over the alimentary canal, liver, stomach and lungs.All organs of the abdominal cavity had become indistinguishable due torampant tumor growth. The mice which had received beads withencapsulated RENCA cells, however showed no hemorrhaging, and only a fewnodules on the alimentary canal. In addition, comparison of test andcontrol groups showed that in the test group, nodules had not progressedbeyond their initial growth under the kidney capsule and beforemacrobead implantation.

EXAMPLE 6

In vitro, freely inoculated RENCA cell growth is inhibited when suchcells are incubated along with macrobead encapsulated RENCA cells. Afurther set of experiments was carried out to determine if this effectwas observable with other cells.

An adenocarcinoma cell line, i.e., MMT (mouse mammary tumor), wasobtained from the American Type Culture Collection. Encapsulated MMTcells were prepared, as described, supra with MMT cells, to producebeads containing 120,000 or 240,000 cells per bead. Followingpreparation of the beads, they were used to determine if they wouldinhibit proliferation of RENCA cells in vitro. Specifically, twosix-well petri plates were prepared, via inoculation with 1×10⁴ RENCAcells per well, in 4 ml of medium. In each plate, three wells served ascontrol, and three as test. One of the three control wells in each platereceived one empty bead. Each of the other wells received either two orthree empty beads. The second set of wells was treated similarly, withwells receiving one, two or three beads containing 120,000 or 240,000MMT cells. Wells were incubated at 37° C. for one week, after whichRENCA cells were trypsinized, washed, and counted, using ahemocytometer. Results are shown in Table 1:

TABLE 1 DISH #1 # of cells retrieved after one week DISH #2 Control # ofcells retrieved after one week (Empty 120,000 Control 240,000 Well#macrobead) MMT cells (Empty Macrobead) MMT cells 1 2.4 × 10⁵ 1.4 × 10⁵2.8 × 10⁵ 1 × 10⁵ 2 2.0 × 10⁵ 1.2 × 10⁵ 3.6 × 10⁵ 7 × 10⁴ 3 4.4 × 10⁵1.25 × 10⁵  2.5 × 10⁵ 9 × 10⁴

EXAMPLE 7

Following the results in example 6, the same experiments was carried outusing 1×104 MMT cells as the inoculant (i.e., the free cells) ratherthan RENCA cells. The experiment was carried out precisely as example 6.Results are set forth in Table 2 below.

TABLE 2 DISH#1 DISH #2 120,000 240,000 Control MMT cells in Control MMTcells in Well# (Empty macrobead) macrobeads (Empty Macrobead) macrobeads1 3.1 × 10⁶ 1.6 × 10⁶ 2.8 × 10⁶ 1.3 × 10⁶ 2 3.3 × 10⁶ 1.0 × 10⁶ 2.6 ×10⁶ 1.1 × 10⁶ 3 3.0 × 10⁶ 6.0 × 10⁵ 2.8 × 10⁶ 5.0 × 10⁵

These results encouraged an in vivo experiment. This is presented inexample 8.

EXAMPLE 8

The mouse mammary tumor cell line (MMT) described supra was used. Usingthe protocols set forth, supra, implants were prepared which contained120,000 cells per bead, and 240,000 cells per bead.

The experimental model used was the mouse model, supra. Twenty-two micewere divided into groups of 4 (control), 9 and 9. The first group, i.e.,the controls, were further divided into three groups: two receivedimplants of one empty bead, one received two empty beads, and onereceived three empty beads.

Within experimental Group A (9 animals), the beads contained 120,000cells, while in experimental Group B, the beads contained 240,000 cells.Within Groups A and B, there were three subdivisions, each of whichcontained three mice. The subgroups received one, two, or three beadscontaining MMT cells.

For the first few days, the mice in Groups A and B were lethargic, withspiky hair. This persisted for about five days, after which normalbehavior was observed. Twenty-one days following implantation, allanimals received injections of 40,000 RENCA cells.

After another twenty days, the control mice exhibited distendedabdomens, and extremely spiky hair. One control mouse died twenty-fivedays following injection, while the remaining control mice appearedterminal. All mice were sacrificed, and tumor development was observed.These observations are recorded in Table 3 infra:

TABLE 3 NUMBER OF EXPERI- MACROBEADS EXPERIMENTAL MENTAL IN MICE CONTROLGROUP A GROUP B 1 ++++ − − 1 ++++ − − 1 + ++ 2 ++++ − − 2 − − 2 ++ ++ 3++++ − − 3 − − 3 − +++

These results show that, of eighteen mice treated, thirteen showed nodisease. Of the mice in Group A, one mouse exhibited a few small nodules(+) and another mouse showed a few tumors(++)

Within Group B, one mouse which bad received one bead, and one mousewhich received two beads showed a few tumors, entangled with intestine.One of the mice which received three beads had developed a large solidtumor and was apparently vezy sick (+++). All control mice bad numeroustumors (++++). The results showed that the encapsulated mouse mammarytumor cells inhibited tumor formation.

EXAMPLE 9

As suggested, supra, the practice of the invention results in theproduction of material which inhibits and/or prevents tumor cellproliferation. This was explored further in the experiment whichfollows.

Additional beads were made, as described supra in example 2, except thatatelocollagen was not included. Hence, these beads are agarose/agarosebeads. RENCA cells, as described, supra, were incorporated into thesebeads, again as described supra.

Two sets of three six-well plates were then used as control andexperimental groups. In the control group, wells were filled with 4 mlsof RPMI complete medium (10% fetal calf serum and 11 ml/1 ofpenicillin). Each control group well was then inoculated with 10,000RENCA cells.

In the experimental group, the RPMI complete medium was conditioned, byadding material secured by incubating ten RENCA containing beads(120,000 cells per bead), in a 35×100 nm petri plate containing 50 ml ofthe RPMI complete medium. Following five days of incubation, medium wascollected from these plates, and 4 mls of it were placed in each testwell. These wells were then inoculated with 10,000 RENCA cells in each.

All plates (both control and experimental) were incubated at 37° C. forfive days. Following the incubation period, cells were trypsinized,washed, pooled, and counted using a hemocytometer. The results are shownin Table 4:

TABLE 4 RENCA CELLS TEST RENCA CELLS WITH CONDITIONED WELL # WITHCONTROL MEDIUM MEDIUM 1 7 × 10⁵   3 × 10⁵ 2 8 × 10⁵ 2.5 × 10⁵ 3 7 × 10⁵3.4 × 10⁵

These results show that the cells, when restricted in, e.g., the beadsof the examples, produced some material which resulted in suppression oftumor cell proliferation.

EXAMPLE 10

The experiment set forth supra showed that RENCA cell growth, inconditioned medium, was about half the growth of the cells in controlmedium. The experiments set forth herein examined whether thesuppression of proliferation would continue after the conditioned mediumwas frozen.

RENCA conditioned medium was prepared by incubating ten RENCA containingbeads for five days. Incubation was in 35×100 mm petri plates, with 50mls RPMI complete medium, at 37° C. Following the incubation, the mediumwas collected and stored at −20° C. Conditioned medium was prepared byincubating MMT (mouse mammary tumor) cell containing beads. The beadscontained 240,000 cell per bead; otherwise all conditions were the same.

Frozen media were thawed at 37° C., and then used in the followingtests. Three six-well plates were used for each treatment, i.e., (i)RPMI control medium, (2) RENCA frozen conditioned medium, and (3) MMTfrozen conditioned medium. A total of 4 mls of medium was dispensed intoeach well. All wells were then inoculated with 10,000 RENCA cells, andincubated at 37° C., for five days. Following incubation, two plates ofsamples were taken from each well, trypsinized, washed, pooled, andcounted in a hemocytometer. At eight days, the remaining three plates ofeach well were tested in the same way.

Results follow:

TABLE 5 FROZEN FROZEN CONTROL CONDITIONED CONDITIONED DISH MEDIUM MEDIUMOF RENCA MEDIUM OF MMT 5 DAYS OLD 1   6 × 10⁵ 5 × 10⁵ 8 × 10⁴ 2 6.8 ×10⁵ 4.2 × 10⁵   8.5 × 10⁴   8 DAYS OLD 3 2.8 × 10⁶ 2 × 10⁶ 8 × 10⁴

When these results are compared to those in example 6, supra, it will beseen that, while the frozen/thawed RENCA conditioned medium did notsuppress proliferation to the same extent that frozen/thawed MMTconditioned medium did (compare examples 6 and 7), it did, nonetheless,suppress proliferation.

EXAMPLE 11

The experiments set forth supra showed that frozen conditioned mediumfrom RENCA- or MMT-containing macrobeads inhibits the proliferation ofRENCA cells in vitro. The experiments set forth herein examined whetherRENCA- or MMT-macrobead conditioned medium, prepared as 30 kd or 50 kdconcentrates by filtration, would inhibit the proliferation of RENCAcells in vitro. The effects of macrobead conditioned media were comparedto the effects of media conditioned in the presence of unrestrictedRENCA and MMT cells growing in monolayer cultures, to determine whetherunrestricted tumor cells grown to confluence also make proliferationregulating material.

For these experiments, 10 macrobeads, each containing 120,000 RENCA orMMT cells (i.e., 1.2×10⁶ cells total) were used to condition the medium(complete RPIMI) over a period of 5 days. In parallel, 1.2×10⁶ RENCA orMMT cells, i.e., the same number of cells, were plated in a culture dishand allowed to proliferate as a monolayer over a period of 4 days incomplete RPMI medium. Medium was then changed, and this medium wascollected twenty-four hours later. The reason for the different lengthof time of exposure of the beads and unrestricted cells was thedifference in cell numbers in the monolayers vs. the beads (3- to 5-fold more cells in the monolayers) at the end of the 5-day period. Inother words, unrestricted cells grew so much more rapidly thanencapsulated cells, that there were 3-5 times more cells.

30 kd and 50 kd filters were used to prepare concentrates of theconditioned media that would, presumably, contain the active material,and would also eliminate toxic metabolic and/or waste materials asconfounding factors in the experiments. These contaminants, which arewell known, are too small to be retained on a 30 kd filter. Filtrateswere also tested, but any interpretation of the results with thismaterial is complicated by the presence of the cellular waste products.A serum-free medium (AIM V) was also used in some experiments to becertain that any effects of serum per se were controlled.

Essentially, conditioned medium was collected, either three to five daysafter the macrobeads had been added to it, or twenty-four hours afternew medium had been added to the unrestricted cells. The medium was thenplaced in a test tube filter with an appropriate filter (either a 30 kdor 50 kd filter), and centrifuged for 90 minutes. Material whichremained on the filter is referred to as the “concentrate,” while thatwhich passes through the filter and collects at the bottom of the tubeis the filtrate.

The results, summarized in Tables 6, 7 and 8 which follow, show thatwhen the conditioned medium resulting from the restricted RENCA cells inthe macrobeads was used, this inhibited RENCA cell proliferation byabout 52% in two separate experiments. The 50 kd concentrate inhibitedproliferation by about 99%/, in both cases, while the 30 kd concentrateinhibited proliferation by about 97%.

TABLE 6 Inhibition of RENCA Cell Growth in RENCA Macrobead ConditionedMedium and Reconstituted Concentrates Unconditioned RENCA Macrobead 30KConcentrate of this 50K Concentrate of this Plate RPMI MediumConditioned Medium Medium Medium Number # of Cells # of Cells Inhibition# of Cells Inhibition # of Cells Inhibition 1  1.6 × 10⁶ 7.5 × 10⁵ 51.3%4.2 × 10⁴ 97% 2.0 × 10⁴ 99% 2 1.65 × 10⁶ 8.0 × 10⁵ 51.5% 5.0 × 10⁴ 97%2.0 × 10⁴ 99%

TABLE 7 Inhibition of RENCA Cell Growth in RENCA Macrobead ConditionedMedium and Reconstituted Concentrates Unconditioned RENCA Cell Culture30K Concentrate of this 50K Concentrate of this Plate Medium ConditionedMedium Medium Medium Number # of Cells # of Cells Inhibition # of CellsInhibition # of Cells Inhibition 1 1.6 × 10⁶ 1.3 × 10⁶ 18.8% 1.1 × 10⁶31.3% 9.0 × 10⁵ 43.8% 2 1.6 × 10⁶ 1.2 × 10⁶ 25.0% 1.0 × 10⁶ 37.5% 9.5 ×10⁵ 40.6%

TABLE 8 Inhibition of RENCA Cell Growth in RENCA Macrobead ConditionedMedium and Concentrate (AIM V Medium) AIM V CONDITIONED 30K 50K PLATECONTROL MEDIUM CONCENTRATE CONCENTRATE NUMBER MEDIUM # cells %inhibition # cells % inbibition # cells % inhibition 1 1.3 × 10⁶ 6.0 ×10⁵ 54% ˜5.0 × 10⁴ 96% ˜4.0 × 10⁴ 97% 2 1.3 × 10⁶ 5.5 × 10⁵ 58% ˜5.0 ×10⁴ 96% ˜4.0 × 10⁴ 97%

An important point of the experiment is that MMT cells and RENCA cells,when entrapped and restricted in the macrobeads both suppress RENCA cellproliferation, indicating that the proliferation restrictive effect isnot specific to tumor type. These experiments confirm those of Example 8in which MMT-containing macrobeads suppressed the proliferation of RENCAcells in vivo. In addition, they extend the findings to indicate thatthe material released from the macrobeads into the medium containsmolecules that are at least 30 kd in molecular weight which areresponsible, in part, for the proliferation-restrictive effect. Finally,these experiments show that the macrobead-restricted RENCA and MMT cellsproduce far more of the proliferation-suppressing material than the samecells grown to confluency in monolayer cultures.

EXAMPLE 12

The experiments set forth above show that both MMT- and RENCA-macrobeadconditioned media contain material released from theproliferation-restricted cells in the macrobead that can inhibit theproliferation of RENCA cells in vivo and in vitro. Importantly, theexperiments show that the proliferation-inhibitory effect is notspecific to tumor type. The experiments set forth herein examine whetherthe effect is also independent of the species in which the tumororiginally arose. Here, the tumor cell proliferation-inhibitory effectsof a human breast cancer-derived cell line on RENCA cells (usingmacrobeads and macrobead-conditioned media) and also MMT cells (usingmacrobead-conditioned media only) in vitro were examined.

The methodologies for these in vitro studies were similar to thosedescribed in the examples above. 100,000 MCF-7 cells, (human breastcancer cells) were encapsulated in macrobeads, and the resulting MCF-7macrobeads were incubated with RENCA cells (10,000 per well) for 5 daysto evaluate the proliferation-inhibitory effects of the macrobeads. Inaddition, MCF-7 macrobead-conditioned medium was prepared over a 5-dayincubation period and tested on both RENCA and MMT cells. Cellproliferation was measured over a 5-day period.

The results are set forth below:

TABLE 9 RESULTS OF MCF-7 MACROBEADS ON RENCA TARGET CELLS CONTROL Well #(Empty Macrobeads) MCF-7 MACROBEADS 1 8.4 × 10⁵ 4.4 × 10⁵ 2 8.0 × 10⁵4.4 × 10⁵ 3 7.4 × 10⁵ 3.8 × 10⁵

TABLE 10 RESULTS OF MCF-7 CONDITIONED MEDIUM ON RENCA TARGET CELLS RPMIConditioned RPMI Control Medium Plate Medium MCF-7 1 9.0 × 10⁵ 5.0 × 10⁵2 8.8 × 10⁵ 4.8 × 10⁵

TABLE 11 RESULTS OF MCF-7 CONDITIONED MEDIUM ON MMT TARGET CELLS RPMIControl RPMI Conditioned Plate Medium Medium:MCF-7 1 5.0 × 10⁵ 1.5 × 10⁵2 6.0 × 10⁵ 1.8 × 10⁵

The results show that MCF-7, a human breast adenocarcinoma cell line,when proliferation-restricted in macrobeads, produces a material thatinhibits the proliferation of mouse renal adenocarcinoma cells and mousebreast cancer tumor cells to a significant degree (30-70%) asdemonstrated by both the macrobeads themselves and conditioned mediaderived therefrom. This indicates that the proliferation-inhibitoryeffect of growth-restricted cancer cells is independent of both tumortype and species of tumor origin, i.e., mouse and human.

EXAMPLE 13

The experiments set forth above demonstrate that a human-derived breastadenocarcinoma cell line (MCF-7), when growth-restricted in macrobeads,produces proliferation inhibition of mouse renal and mouse breastadenocarcinoma cells in vitro. The experiments set forth herein examinewhether a parallel effect of MCF-7-containing macrobeads on RENCA celltumor growth in vivo exists.

Eighteen Balb/c mice were injected with 20,000 RENCA cellsintraperitoneally. After three days the mice were divided into twogroups. Group 1 had six mice and Group 2 had the remaining twelve mice.Group 1 mice, the controls, were transplanted with three emptymacrobeads each. Group 2 mice each received three MCF-7-containingmacrobeads (100,000 cells per bead). After twenty-five days, 2 mice fromGroup 1and three mice from Group 2 were sacrificed. The same number weresacrificed on day twenty-six and the remaining mice were sacrificed onday twenty-seven.

On necroscopy, the peritoneal cavities of the control mice were observedto be completely packed with tumor, and the normal organs were difficultto identify. We classified this as ++++(100%) tumor intensity. In thetreated mice, tumor intensity was rated at +(10-20%).

These results show that macrobeads containing human breastadenocarcinoma cells are capable of inhibiting renal cell adenocarcinomatumor growth in mice, confirming again that the cancer-cellproliferation/tumor growth-inhibitory effect is neither type-specificnor species-specific.

EXAMPLE 14

The experiments set forth above demonstrate that the cellproliferation/tumor growth inhibitory effect of macrobeadgrowth-restricted tumors is neither tumor-type nor species specific. Theexperiments set forth herein examine whether (macrobead)proliferation-restricted mouse breast adenocarcinoma cells can inhibitthe growth of both spontaneous mammary tumors and tumors resulting fromthe injection of MMT cells.

C3H mice have a very high incidence of the development of mammary tumorsover their life span. Seven mice at risk for the development of suchtumors showed tumors at sixteen months of age. At this time, five of theseven mice were implanted with four MMT macrobeads containing 100,000cells each. The remaining two control mice received four emptymacrobeads each. The two control mice developed large tumors and diedwithin three months after the bead implants. The treated mice weresacrificed eleven months after the MMT macrobead implants. The retrievedmacrobeads, organs and tumors were examined grossly and histologically.Hemotoxylin & Eosin staining of the MMT macrobeads showed viable cells.The pre-existing tumors had not increased in size, and there was noevidence of any new tumor development.

Experiments in which MMT tumor cells were injected subcutaneously in thethoracic region were also performed. Fourteen C3H mice were divided intotwo groups. The five control group mice were implanted with three emptymacrobeads each. The nine treated mice received three MMT-containingmacrobeads (240,000 cells) each. Three weeks after implantation allfourteen mice were injected subcutaneously in the mammary area with20,000 MMT cells each.

Within twenty-five to thirty days, the five control group mice becameill with evident tumor formation, and all were dead by thirty-five dayspost-injection. The nine treated mice, observed weekly, continuedwithout any evidence of tumor formation or ill health during thisperiod. Ten to twelve months after tumor injection, four of the ninetreated mice developed lumps and lost their fur in patches. Theremaining five mice were implanted again with three MMT macrobeadsthirteen months after the initial tumor injection. One mouse died threedays after this surgery, but on necropsy was completely free of tumor.The four surviving mice were sacrificed eight months after the secondmacrobead implant. Necropsy showed minimal or no tumor proliferation.

An additional observation from these experiments was that the beadsretrieved from the first implantation contained viable tumor cells basedboth on histology and their ability to resume aggressive tumor growthpatterns in tissue culture after removal from the bead.

The results of these experiments show that the cell proliferation/tumorgrowth-inhibiting effects of macrobead-restricted cancer cells, in thiscase mouse mammary adenocarcinoma cells, can influence the developmentand growth of both spontaneously arising tumors and experimentallyinduced tumors arising from the injection of tumor cells into themammary area.

EXAMPLE 15

The experiments set forth above demonstrate a tumor cellproliferation/tumor growth-inhibitory effect of macrobeadproliferation-restricted cancer cells that is characterized by itseffectiveness across tumor types and across species, as well as in bothspontaneous and artificially-induced tumors. The experiments describedherein extend these findings to examine the effects ofmacrobead-entrapped, proliferation-restricted human prostateadenocarcinoma-derived cells (ARCap10), mouse (Balb/c) renaladenocarcinoma cells (RENCA cells), and mouse (C3H) mammaryadenocarcinoma cells (MMT) on the proliferation of ARCaP10 tumor cellsand ARCaP10 tumor growth in nude (Nu/Nu) mice.

In the first series of experiments, fifteen Nu/Nu mice were injectedwith 2.5×10⁶ ARCaP10 cells subcutaneously in the flank. On the twentiethday after injection, at which time the average maximal tumor diameterwas 0.5 cm, the mice were divided into two groups. Nine were implantedwith four ARCAP10 macrobeads (1.0×10⁵cells per macrobead) each, and sixcontrol mice received four empty macrobeads each.

Ten weeks after implantation, five of the control mice had very largevascularized tumors (average 2.5 cm in diameter) and one mouse showed aslightly smaller tumor (less than 0.5 cm). In the treated group, fivemice showed complete regression of the initial tumors, and all remainedtumor free until sacrifice at eight months. Two mice showed no tumorgrowth, i.e., their tumors had the same maximal diameter as they had hadat the time of implantation of the macrobeads, and two mice showedtumors that had enlarged since implantation of the macrobeads.

The results (tumor volume and size (1×w×h)) of an experiment in whichRENCA-containing macrobeads (1.2×10⁵) were implanted eighteen days aftersubcutaneous flank injection of 3.0×10⁶ ARCAP10 tumor cells per animalin 4 Nu/Nu mice are set forth below:

TABLE 12 SIZE OF TUMORS OBSERVED IN TREATED MICE (in mm) 10 Days 14 DaysTreated 3 Days Before Day of 3 Days After 6 Days After After After MouseTransplant Transplant Transplant Transplant Transplant Transplant Number(3/3/98) (3/6/98) (3/9/98) (3/12/98) (3/16/98) (3/20/98) 1 3.5 × 3 ×flat 6.2 × 5.4 × flat 4 × 4 × flat disappearing 0 0 2 3 × 3 × 1.5 5.1 ×2.2 × 2 4 × 2 × 0.5 3 × 3 × 0.4 2 × 2 × 0.3 2 × 2 × 0.3 3 3 × 2.5 × 13.1 × 3.3 × 1 3 × 2 × 0.5 3 × 2 × 0.2 3 × 2 × 0.2 3 × 2 × 0.2 4 2.5 ×2.5 × flat 3.2 × 3.4 × 0.5 speck under skin 0 0 0

TABLE 13 VOLUME OF TUMORS OBSERVED IN TREATED MICE 3 Days Before Day of3 Days After 6 Days After 10 Days After 14 Days After Treated MouseTransplant Transplant Transplant Transplant Transplant Transplant Number(3/3/98) (3/6/98) (3/9/98) (3/12/98) (3/16/98) (3/20/98) 1 2.76 8.811.68 0 0 0 2 7.10 11.81 2.10 1.89 0.63 0.63 3 3.95 5.38 1.58 0.63 0.630.63 4 1.64 2.86 0 0 0 0

In another experiment110Nu/Nu mice were injected with 2.5×10⁶ APCaP10cells, with six of the mice showing tumor development sixty-four daysafter injection. Three of these mice were given four MMT macrobeads(2.4×10⁵ cells) each and three received empty macrobeads. The resultsare set forth below:

TABLE 14 SIZE OF TUMORS OBSERVED IN TREATED MICE (in mm) 5 Days BeforeDay of 18 Days After 22 Days After 27 Days After 30 Days After TreatedMouse Transplant Transplant Transplant Transplant Transplant TransplantNumber (2/5/98) (2/10/98) (2/28/98) (3/4/98) (3/9/98) (3/12/98) 1 2 × 2× 1 3 × 3 × 1.5 1 × 1 × 0.5 0 0 0 2 3 × 2 × 1 3 × 2.5 × 1 2 × 2 × flat<1 mm <0.8 mm <0.8 mm 3 4 × 4 × 1.5 6 × 6 × 1.5 6 × 2 × flat 4 × 1 ×flat 3 × 1 × flat 3 × 1 × flat

TABLE 15 SIZE OF TUMORS OBSERVED IN CONTROL MICE (in mm) Control 5 DaysBefore 18 Days After 22 Days Atter 27 Days After 30 Days After MouseTransplant Day of Transplant Transplant Transplant Transplant TransplantNumber (2/5/98) (2/10/98) (2/28/98) (3/4/98) (3/9/98) (3/12/98) 1 4 × 4× 1.5 5 × 5 × 2 6.5 × 6 × 3 6.5 × 6 × 3 6.5 × 6 × 3 7 × 7 × 3 2 3 × 2 ×1 4 × 6 × 3 4.5 × 7 × 3 5 × 8 × 3 11 × 12 × 5 13.3 × 13.3 × 6.5 2^(nd)tumor: 6 × 6 × 1 3 5 × 4 × 1 5 × 4 × 2 5 × 4.6 × 2.5 5 × 5 × 2.5 6 × 6 ×2.5 7 × 7 × 2.5 (multilobe) 2^(nd) tumor: 2^(nd) tumor: 2 × 2 × 1 3 × 3× 0.5

The results of these experiments further confirm the cross-species,cross-tumor nature of the tumor growth-inhibiting effect ofproliferation restriction on tumors of various types. In addition, theseexperiments demonstrate the ability of the proliferation-restrictedcancer cells not only to suppress tumor growth and to prevent tumorformation, but also to cause actual regression of in vivo tumors.

EXAMPLE 16

The experiments set forth above showed that proliferation-restrictedcancer cells from several types of tumors and species can inhibit theproliferation of the same and different cancer cell types in vitro andprevent the formation of both spontaneous and induced tumors, preventthe growth of tumors, and cause tumors to regress in vivo in an effectthat is independent of species and cancer type. The experiment set forthherein describes the extension of the findings to another species(rabbit) and a rabbit tumor known to have been induced virally (VX2).

In this experiment, a New Zealand White Rabbit (2.5 lbs.) was injectedintramuscularly in one thigh (two sites) with 0.5 ml of a VX2 tumorslurry (characterized as being able to pass through a #26 gauge needle)at each site. At 3.5 weeks, a 5 cm×2.5 cm (1xw) tumor had appeared onthe dorsal thigh and two 3 cm-diameter tumors were present on theventral thigh. At this point, 211 macrobeads (108 RENCA cell beads, 63MMT cell beads, and 40 MCP-7 human breast cancer cell-containing beads)were implanted intraperitoneally. Within two days, the tumor on thedorsal thigh had shrunk by approximately 50%; however, the two ventraltumors did not change. The animal was sacrificed ten days aftermacrobead implantation. On necropsy, there was a clear differencebetween the dorsal and ventral tumors in that the former was muchsmaller than it had been at the time of macrobead implantation, whereasthe two ventral tumors were both hemorrhagic and necrotic.

This experiment extends the findings of the effectiveness ofproliferation restriction of various types of cancer cells in relationto the prevention, arrest, and even regression of tumor growth toanother species, the rabbit, adds a tumor of known viral origin to thelist of cancer types, and further supports the cross-tumor andcross-species nature of the growth inhibiting effect, since acombination of mouse renal, mouse breast and human breast cancercell-containing macrobeads were used. In addition, the experiment adds alarger animal model to the in vivo testing of the effectiveness ofproliferation-restriction of cancer cells for the treatment of cancer.

EXAMPLE 17

The experiments set forth above show that proliferation-restriction ofvarious types of tumor cells results in their ability to inhibit thegrowth of cells of the same or different type in vitro and to preventthe formation of, suppress the growth of, or cause regression of varioustypes of tumors in vivo and that the effects seen are independent oftumor type and species. The experiments set forth herein evaluated thelong-term viability of the proliferation-restricted RENCA cancer cellsin agarose-agarose macrobeads maintained in culture over periods of 1month, 6 months, 2 years, and 3 years using histological, culture, andin vivo techniques. MMT-containing macrobeads were maintained in culturefor up to six months. In addition, RENCA- and MMT-containing macrobeadsretrieved from Balb/c and C3H mice respectively after periods of 2 to 8months after implantation were examined for viable tumor cells by bothhistological and culture techniques.

For these experiments the agarose-agarose macrobeads were prepared witheither 1.2×10⁵ RENCA cells or 2.4×10⁵ MMT cells. They were examinedhistologically (hermatoxylin & eosin staining) and by culture techniquesfor cell viability and tumor characteristics at the intervals describedsupra. For the RENCA macrobeads, cell numbers increased approximately 3-to 5-fold over the first month with a subsequent additional doubling insix months. After one year, there was a continued increase in cellularmass, but the rate of cell proliferation had decreased. After two years,amorphous material had begun to appear in the center of the bead, andthe cell mass/numbers did not appear to be increasing, although mitoticfigures were still evident. After three years, there appeared to besomewhat more amorphous material in the center of the bead, but the cellmass/number was stable. MMT macrobeads have been followed for only sixmonths, but the early pattern of cell proliferation and bead appearanceis similar to that of RENCA.

For evaluation of the viability and biological behavior of the RENCA andMMT cells at the intervals described above, ten beads were crushed andplated in two or more 25 cm² tissue culture flasks in complete RPMImedium. The flasks were then observed for cell growth. At one and sixmonth intervals, the number of viable cells retrievable from the beadsincreased. At one year, the number of RENCA cells growing from thecrushed bead appeared to be similar to that at six months. At two andthree years, the proportion of viable cells appears to be somewhat less,dropping to approximately 20% of the maximum number they reached in thebead (i.e., in their restricted state) after three years in culture.

For the evaluation of the retrieved RENCA and MMT macrobeads after invivo implantation (periods of 1-4 years for RENCA macrobeads and up to 8months for MMT macrobeads), histological techniques have been utilizedto date. The patterns of cell proliferation and mass are very similar tothose of the beads maintained in culture for the corresponding periodsof time, i.e., the cells increase in number at least up to 4 months forRENCA and 8 months for MMT.

For the other cancer cell lines used, such as MCF-7 and ARCAP10, theviability patterns in macrobeads are similar to those observed for RENCAand MMT.

These experiments show that cancer cells can be maintained in vitro forperiods of up to 3 years and in vivo for periods of at least 8 months ina proliferation-restricting environment and that they maintain theirviability for these periods with clear demonstration of increasing cellnumbers up to at least one year. This is important not only for theability to create and to store cancer treatment materials, but also forthe ability of the proliferation-restricted cells to put out tumorgrowth suppressing material in warm-blooded animals over the continuous,prolonged periods likely to be necessary for the successful treatment ofexperimental or naturally-occurring cancer.

EXAMPLE 18

The experiments set forth above show that cancer cells of various typescan be maintained under proliferation-restricted conditions for longperiods of time (up to 3 years) with retention of their ability toproliferate, to form tumors, and to releasecell-proliferation-inhibiting and tumor-growth preventing, suppressing,and even regressive materials. The experiments set forth herein evaluatethe possible toxicity of long-term (one-year) implants of cancercell-containing, agarose-agarose macrobeads in Balbic mice.

Seven Balb/c mice were implanted with 3 RENCA macrobeads each (1.2×10⁵cells per bead). Immediately after surgery the mice appeared ill (spikyfur and lethargy) for a few days, but became healthy again after this.All mice survived in apparent good health for a period of at least oneyear, with one mouse dying of old age and another of unrelated causes.All mice were sacrificed. On necropsy, no abnormalities, such asfibrosis, peritonitis, or tumor growth were observed. All organsobserved appeared normal, although some adherence of the beads to theserosal surfaces of the intestines were observed, especially where therewere intestinal loops. No interference with the normal function orstructure of the intestines has been observed.

These results show that cancer cell-containing agarose-agarosemacrobeads are well tolerated in experimental animals over a one-yearperiod. These findings show that the proliferation-restrictingcancer-cell beads can be utilized in vivo for the prevention,suppression and regression of cell proliferation, including in vivotumors of various types.

The foregoing examples describe the invention, which includes, interalia, compositions of matter which can be used to produce material whichsuppresses or controls the proliferation of rapidly proliferating cells,particularly suppression of cancer cells. These compositions comprisecells in a log-phase growth (rapid proliferative phase) entrapped in aselectively-permeable material to form a structure which restricts theproliferation of the entrapped cells. As a result of their beingrestricted, the cells produce unexpectedly high amounts of materialwhich suppresses proliferation of rapidly proliferating cells, such ascancer cells. The restricted cells produce more of the material thancomparable amounts of non-restricted cells.

The matter used to make the structures of the invention include anybiocompatible matter which restricts the growth of rapidly proliferatingcells, thereby inducing them to produce greater amounts of cellproliferation/growth-suppressing material. The structure has a suitablepore size such that the above suppressive material can diffuse to theexternal environment, and prevent products or cells from the immunesystem of the host from entering the structure and causing the rejectionof the cancer cells or otherwise impair their ability to survive andcontinue to produce the desired material. The matter used to form thestructure will also be capable of maintaining viable(proliferation-restricted, but surviving) cells both in vitro and invivo, preferably for periods of up to several years by providing for theentrance of proper nutrients, the elimination of cellular wasteproducts, and a compatible physico-chemical intra-structuralenvironment. The matter used to prepare the structure is preferably welltolerated when implanted in vivo, most preferably for the entireduration of implantation in the host.

A non-limiting list of materials and combinations of materials thatmight be utilized includes alginate-poly-(L-lysine);alginate-poly-(L-lysine)-alginate;alginate-poly-(L-lysine)-polyethyleneimine; chitosan-alginate;polyhydroxylethyl-methacrylate-methyl methacrylate;carbonylmethylcellulose; K-carrageenan; chitosan;agarose-polyethersulphone-hexadi-methirine-bromide (Polybrene);ethyl-cellulose; silica gels; and combinations thereof.

The structures which comprise the compositions of matter may take manyshapes, such as a bead, a sphere, a cylinder, a capsule, a sheet or anyother shape which is suitable for implantation in a subject, and/orculture in an in vitro milieu. The size of the structure can vary,depending upon its eventual use, as will be clear to the skilledartisan.

The structures of the invention are selectively permeable, such thatnutrients may enter the structure, and so that theproliferation-inhibiting material as well as cellular waste may leavethe structure. For in vivo use, it is preferred that the structuresprevent the entry of products or cells of the immune system of a hostwhich would cause the rejection of the restricted cells, or otherwiseimpair the ability of the cells to produce the proliferation-suppressivematerial.

Another aspect of the invention includes compositions which are usefulin suppressing cancer cell proliferation. These compositions areprepared by culturing restricted cells as described supra in anappropriate culture medium, followed by recovery of the resultantconditioned medium. Concentrates can then be formed from the conditionedmedium, e.g., by separating fractions having molecular weights ofgreater than 30 kd or greater than 50 kd, which have highanti-proliferative effects on cancer cells.

The invention is not limited to any particular type of proliferativecell. Any rapidly proliferating cell as defined supra may be used inaccordance with the invention including, but not limited to neoplasticcells, embryonic cells, stein cells, cells in a regenerative stage suchas post-injury or trauma, particularly hepatocytes, fibroblasts andepithelial cells. Cancer cells are one preferred type of cell, andexemplary types of cancer cells which may be used include renal cancercells, mammary cancer cells, prostate cancer cells, choriocarcinomacells and so forth. The cancer cells may be of epithelial, mesothelial,endothelial or germ cell origin, and include cancer cells that generallydo not form solid tumors such as leukemia cells.

As will be clear from this disclosure, a further aspect of the inventionis therapeutic methods for treating individuals suffering fromhyperproliferative diseases such as cancer, inflammation, fibrosis, andtrophoblastosis. It is also an aspect of the invention to control thegrowth of cells in a proliferative phase so that they reproduce in acontrolled manner. This is particularly useful, for example, incontrolling the production of keloids and scar tissue, preventingadhesions after abdominal surgery, and possibly in hyperproliferativeskin diseases such as psoriasis. When used in a therapeutic context, aswill be elaborated upon infra, the type of cell restricted in thestructure need not be the same type of cell causing (or which may cause)the patient's suffering, although it can be. One such method involvesinserting at least one of the structures of the invention into thesubject, in an amount sufficient to cause suppression of cellproliferation in the subject. Preferably the method is for the treatmentof cancer and the subject is a human being, although it is applicable toother animals, such as domestic animals, farm animals, or any type ofanimal which suffers from cancer.

The composition of the present invention can be used as primary therapyin the treatment of hyperproliferative diseases, and as an adjuncttreatment in combination with other therapies. For example, patients maybe treated with compositions and methods described herein, inconjunction with known therapies such as radiation therapy,chemotherapy, treatment with other biologically active materials such ascytokines, anti-sense molecules, steroid hormones, gene therapy, and thelike. The compositions and methods of the invention can also be used inconjunction with surgical procedures to treat cancer, e.g., byimplanting the macrobeads after resection of a tumor to prevent regrowthand metastases. Cancers which are present in an inoperable state may berendered operable by treatment with the anti-proliferative compositionsof the invention.

The compositions of the invention can also be used prophylactically inindividuals at risk for developing cancer, e.g., presence of individualrisk factors, family history of cancer generally, family history ofcancer of a specific type (e.g. breast cancer), and exposure tooccupational or other carcinogens or cancer promoting agents. Forprophylaxis against cancer, a prophylactically effective amount of thestructures of the invention are administered to the individual uponidentification of one or more risk factors.

As indicated by the examples, supra, the antiproliferative effect is notlimited by the type of proliferative cell used, nor by the species fromwhich the proliferative cell originated. Hence, one can administerstructures which contain, e.g, cancer cells of a first type to a subjectwith a second, different type of cancer. Further, proliferative cells ofa species different from the species being treated can be used in theadministered structures. For example, mouse cancer cells may berestricted in the structures of the invention, and then be administeredto a human. Of course, the structures may contain cancer cells from thesame species as is being treated. Still further, the proliferative cellsmay be taken from the individual to be treated, entrapped andrestricted, and then administered to the same individual. In otherwords, the patient's own cancer cells may be restricted and used tosuppress the same cancer.

Yet another aspect of the invention is the use of concentrates, asdescribed herein, as therapeutic agents. These concentrates may beprepared as described herein, and then be administered to a subject witha hyperproliferative disease such as cancer, or to a patient in need ofcontrolling the rate of cell growth, such as a post-op patient having awound in a site where excessive scar tissue would be detrimental or issimply not desired. All of the embodiments described supra may be usedin preparing the concentrates. For example, following in vitro cultureof structures containing mouse cancer cells, concentrates can beprepared and then administered to humans. Similarly, the structures cancontain human cells, and even cells from the same individual. Also, asdiscussed supra, the type of cancer cell used to prepare the concentratemay be, but need not be, the same type of cancer as the subject suffersfrom. Hence, murine mammary cancer cells may be used, e.g., to prepare aconcentrate to be used to treat a human with melanoma, or an individualwith prostate cancer may have some of his prostate cancer cells removed,entrapped in a structure of the invention, cultured in an appropriatemedium, and then have resulting conditioned medium filtered to produce aconcentrate. It should be borne in mind that the conditioned mediaresulting from in vitro cultures of the structures of the invention isalso a part of the invention.

Processes for making the structures of the invention, as well as theconcentrates of the invention, are also a part of the invention. In thecase of the concentrates, one simply cultures the structures of theinvention for a time sufficient to produce a sufficient amount ofantiproliferative material and then separates the desired portions fromthe resultant conditioned medium, e.g., by filtration with a filterhaving an appropriate cut off point, such as 30 kilodaltons or 50kilodaltons.

Other facets of the invention will be clear to the skilled artisan, andneed not be set out here.

The terms and expression which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expression of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

We claim:
 1. Composition useful in suppressing proliferation of cancercells, produced by entrapping cancer cells in a biocompatible,selectively-permeable structure, culturing the entrapped cancer cells ina culture medium to restrict proliferation of said entrapped cancercells, wherein growth of said cancer cells is restricted by entrapmentin said structure and said restricted cancer cells produce a materialwhich suppresses proliferation of cancer cells, and filtering the mediumthrough a filter which separates material having a molecular weight ofat least about 30 kd from material having a molecular weight of lessthan 30 kd and recovering the separated material having a molecularweight of at least about 30 kd as measured by said filtering to providesaid composition.
 2. The composition of claim 1, wherein said entrappedcancer cells are epithelial cells.
 3. The composition of claim 1,wherein said entrapped cells are selected from the group consisting ofbreast cancer cells, renal cancer cells, prostate cancer cells andchoriocarcinoma cells.
 4. The composition of claim 1, wherein saidentrapped cells are human cells.
 5. The composition of claim 1, whereinsaid entrapped cells are mouse cells.
 6. The composition of claim 1,wherein said structure contains from about 10,000 to about 500,000cells.
 7. The composition of claim 1, wherein said structure containsfrom about 30,000 to about 250,000 cells.
 8. A method for suppressingproliferation of cancer cells in a subject in need thereof, comprisingadministering to said subject a sufficient amount of the composition ofclaim 1 to suppress proliferation of cancer cells in said subject. 9.The method of claim 8, wherein said subject is a human.
 10. The methodof claim 8, wherein said entrapped cancer cells are of epithelialorigin.
 11. The method of claim 8, wherein said entrapped cells areselected from the group consisting of renal cancer, choriocarcinoma,breast cancer, and prostate cancer.
 12. The method of claim 8, whereinsaid entrapped cancer cells are human cancer cells.
 13. The method ofclaim 8, wherein said entrapped cancer cells are mouse cancer cells. 14.The method of claim 8, wherein said entrapped cells are of the same typeof cancer cell being suppresses in said subject.
 15. The method of claim8, wherein said entrapped cells are cells taken from the subject towhich said composition is administered.
 16. The method of claim 8,wherein said structure contains from about 10,000 to about 500,000cells.
 17. The method of claim 16, wherein said structure contains fromabout 30,000 to about 250,000 cells.
 18. A process for producing amaterial which has a cancer cell proliferation-inhibiting effect,comprising entrapping cancer cells in a biocompatible,selectively-permeable structure, culturing the entrapped cancer cells ina culture medium to restrict proliferation of said entrapped cancercells, wherein growth of said cancer cells is restricted by entrapmentin said structure and said restricted cancer cells produce a materialwhich suppresses proliferation of cancer cells, and filtering the mediumthrough a filter which separates material having a molecular weight ofat least about 30 kd from material having a molecular weight of lessthan 30 kd and recovering the separated material having a molecularweight of at least about 30 kd as measured by said filtering to providesaid material.
 19. The process of claim 18, wherein said entrappedcancer cells are epithelial cells.
 20. The process of claim 18, whereinsaid cancer cells are s elected from the group consisting of breastcancer cells, renal cancer cells, prostate cancer cells,andchoriocarcinoma cells.
 21. The process of claim 18, wherein said mediumis serum free.
 22. The process of claim 18, wherein said entrappedcancer cells are human cells.
 23. The process of claim 18, wherein saidentrapped cancer cells are mouse cells.
 24. The process of claim 18,wherein said structure contains from about 10,000 to about 500,000cancer cells.
 25. The process of claim 24, wherein said structurecontains from about 30,000 to about 250,000 cancer cells.
 26. Theprocess of claim 18, wherein said structure is a bead.